Updated project metadata. Membrane vesicles (MVs) are produced by species across all domains of life and have diverse physiological functions and promising applications. While the mechanisms for vesiculation in Gram-negative bacteria are well-established, the genetic determinant and regulation of MVs biogenesis in Gram-positive bacteria remain still largely unknown. Here, we demonstrate that a Q225P substitution in the alternative sigma factor SigB greatly triggers MVs production in Staphylococcus aureus strain Newman by directly repressing expression of alkaline shock protein 23, wherein the Q225P mutation hinders the specific binding of SigB to the asp23 promoter. Isogenic deletion of asp23 consistently promotes MVs formation in Newman, highlighting the critical role of both sigB and asp23 in modulating S. aureus vesiculation. While bacterial growth and cytoplasmic membrane fluidity do not be impaired, mutation of asp23 elicits a weakened cell wall and enhanced autolysis that potentially involved in LrgAB-controlled hydrolases and PSMα-mediated activities, which ultimately leads to hypervesiculation in Newman. Furthermore, TEM and proteomic analysis suggest nearly identical morphology and composition, but virulence-associated factors are significantly enriched in MVs upon asp23 deletion. Overall, this study reveals novel genetic determinants and cues underlying S. aureus vesiculation, advancing the understanding of the physiology of MVs biogenesis in Gram-positive bacteria.